1. Field of the Invention
This invention relates to the discovery of new phosphorylated, polyanionic peptides that are surprisingly powerful inhibitors of mineral formation, particularly the crystallization of calcium carbonate and calcium phosphate. The molecules may be useful in a variety of applications including but not limited to tartar control, prevention of industrial scaling, use as dispersants, corrosion inhibition, prevention of pathologic calcification, and control of biofouling.
2. Discussion of Background
Biological mineralization is a fundamental process in nature. Formation of bones and teeth from calcium phosphate and formation of shells and reefs from calcium carbonate are but two examples of this process.
Unfortunately, mineral deposits also frequently occur where they are not wanted. In the body, mineral deposition may contribute to dental plaque, hardening of the arteries, various organ stones, and failure of prosthetic devices like implanted heart valves. In the marine environment, the biomineral structures may cause problems as in the case of barnacles growing on the hulls of ships, adding extra bulk and creating drag. In industry, mineral scale forms on surfaces of cooling towers and other devices preventing their proper operation as heat exchangers and frequently promoting localized corrosion.
Because of the problems associated with unwanted mineral deposition, much effort has been devoted to finding mineralization inhibitors, particularly in industry, that might be used to prevent harmful mineral formation.
Molecules for prevention of mineral deposition have ranged from simple ions like Mg.sup.+2 (Pytkowicz, R.M., J. Geol. 73, 196-199 (1965)) and PO.sub.4.sup.3- or pyrophosphate (Simkiss, K., Biol. Rev. 39, 487-505 (1964)) to more complex organic materials. Inhibition by simple ions is based on the ability of these ions to interfere with the orderly formation of the crystalline lattice of the mineral, such as CaCO.sub.3. In addition, phosphate and polyphosphates have the property of protecting metallic surfaces by forming very thin films that cover potential sites of corrosion on the surfaces.
Phosphonates were introduced in the late 1960's (Ralston, U.S. Pat. No. 3,336,221 (1967)). These are small organic molecules with PO.sub.3 groups attached directly to a central carbon atom via a covalent bond to phosphorus. The phosphonates are very effective inhibitors of crystallization that work by adsorbing to crystal surfaces. Hydroxyethylidene diphosphonate (HEDP) is perhaps the most widely used phosphonate, still among the most powerful inhibitors of CaCO.sub.3 formation known.
Use of phosphonates has some disadvantages though. For example, phosphonates can be degraded during chlorination which in turn may lead to elevated phosphates and associated phosphate scales. Phosphonates themselves may also precipitate under common operating conditions. HEDP is an exceptionally effective inhibitor of crystal nucleation on a weight basis as shown by its effect on lengthening the induction period prior to crystal growth, but it is not at all effective at inhibiting crystal growth after it begins, (Sikes and Wheeler, CHEMTECH 1988, pp. 620-626 (1988).)
More recently, as a result of continuing efforts to identify better inhibitors, polyacrylat and other polyanionic materials have been identified (Rohm and Haas Company, Technical Bulletin CS-513A (1985), Fong and Kowalski, U.S. Pat. No. 4,546,156 (1985)). In the 1980's, antiscaling and anticorrosion technology has been based increasingly on use of synthetic polymers under alkaline conditions. The current trend in synthetic polymers for water treatment is the use of random copolymers or terpolymers with alternating side groups of COO.sup.- with groups like OH, CH.sub.3, PO.sub.3.sup.2-, SO.sub.3.sup.2- etc.
Among the newest approaches to developing mineral deposition inhibitors involves naturally-occurring proteins and polysaccharides that regulate mineral formation by organisms (U.S. Pat. Nos. 4,534,881 (1985), 4,585,560 (1986); 4,603,006 (1986); and 4,587,021 (1986) by C.S. Sikes and A.P. Wheeler). This approach led to the identification of a new class of polyanionic/hydrophobic peptides that are even more powerful inhibitors of crystallization on a weight basis than the natural protein (U.S. application Ser. No. 07/088,247).
In spite of the above approaches to solving the problems of unwanted mineral deposition, there remains a strong need for new and more powerful inhibitors of mineral deposition which could be used in the body, in a marine environment, or industrially, etc.
Now the inventor has discovered that certain phosphorylated, polyanionic peptides are unexpectedly much more effective inhibitors, on a weight basis, than other similar peptides. They are particularly effective inhibitors of calcium phosphate formation, but also of calcium carbonate formation and other mineral depositions as well.
A clue to the identity of the new inhibitors was taken from the report that certain protein inhibitors of calcium deposition from saliva had two phosphoserine residues adjacent to an N-terminal aspartic acid residue (Hay, D.I., et al., Inorg.Persp.Biol.Med.2, 271-285 (1979)). This idea was then contemplated along with the concept that polyelectrolytic polypeptides may adsorb to surfaces by binding only by a few monomers at one end of the molecule, with the rest of the molecule suspended unbound from the surface (Juriaanse et al., J. of Coll. and Interface Sci. 76, 212-219 (1980)). The inventor explored the idea that the terminal residues may be critical in binding of peptides to crystals. That phosphoserine itself is involved in some way in inhibitory activity of proteins and peptides is suggested by Schlesinger et al., in Chemical Aspects of Regulation of Mineralization, C.S. Sikes and A. P. Wheeler, eds., 33-38, 1988 and A. P. Wheeler and C. S. Sikes, in Chemical Perspectives on Biological Mineralization, S. Mann et al., eds., in press 1989.
In short, the present invention identifies new polyanionic polypeptide molecules. These materials preferably contain one to three phosphoserine residues on one end of the molecule. The rest of the molecule is preferably composed of aspartic acid residues. Sulfated, phosphonated, and sulfonated derivatives of serine and other amino acids are also contemplated for inclusion at one of the terminal regions of the molecules.
The N-terminally phosphorylated polyanionic polypeptides of this invention have not been specifically described before. Such a molecule appears to fall broadly within the claims of U.S. Pat. No. 4,534,881 (Sikes and Wheeler 1985). However, the present molecules were not specifically identified therein and there was no suggestion of any special activity inherent in them.
Molecules of H--(Ala).sub.m --(Asp).sub.n --(pSer).sub.x --OH where m =2-10, n=10-60, and x=2-5 were claimed in U.S. application Ser. No. 07/088,247 (Sikes and Wheeler, 1988). However, these molecules contain hydrophobic clusters, unlike the molecules herein. In addition, there was no mention therein of a molecule containing only one N-terminal phosphoserine residue, as in this invention.